Chapter 16 Flow Cytometry: A New Tool in Brewing Technology K.J. Hutter, K.J. HutterSearch for more papers by this authorC. Lange, C. LangeSearch for more papers by this author K.J. Hutter, K.J. HutterSearch for more papers by this authorC. Lange, C. LangeSearch for more papers by this author Book Editor(s):Katherine Smart, Katherine Smart Oxford Brookes University, Oxford, UKSearch for more papers by this author First published: 19 January 2003 https://doi.org/10.1002/9780470696040.ch16 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains section titled: Introduction Materials and methods Results and discussion Acknowledgement References Brewing Yeast Fermentation Performance, Second Edition RelatedInformation

Using flow cytometric analysis, the rapid detection of glycogen content of yeast cells in process is possible. Glycogen is a sensitive parameter which can express the physiological state of the yeast cells. A procedure was developed to stain and measure glycogen in yeast cells using flow cytometry. Glycogen content of lager yeast cells was tested after 1 and 15 It of nutrient limitation.

With flow cytometric cell cycle analysis. fermentation processes become transparent. This implies that we can control the process, by determining growth phases of the yeast population at specific times during the fermentation process. and that the process can be optimised and regulated directly.

The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells. (C) 2000 Wiley-Liss, Inc.

Une levure de qualite elevee s'illustre par une activite de multiplication elevee pendant la propagation et a l'entonnement (forte replication = taux eleve de cellules en bourgeonnement qui se trouvent en phase G 2 du cycle cellulaire) et egalement par un pouvoir fermentaire eleve pendant la fermentation (taux eleve de cellules de levure qui se trouvent en phase G 1 du cycle cellulaire). Ces proprietes de la levure peuvent etre induites et stabilisees par une exploitation optimale des equipements techniques de chaque brasserie ou par un changement de la conduite du procede (par ex. succession de brassins, temps d'intervalle du Darauflassen, quantite de mout fin cuisson). La connaissance des parametres physiologiques permet l'etablissement de strategies de conduite qui aboutit a une optimisation du procede de fabrication.

The glycolytic pathway inhibitor 2-deoxyglucose (2-DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18(HPV 18) in cervical carcinoma cells and derived non-tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down-regulation is selective, since other reference genes are not affected or even up-regulated under the same experimental conditions. Moreover, 2-DG appears to restore the normal half-life of the tumor suppressor gene product p53, because the protein is strongly up-regulated after HPV18 E6/E7 suppression. The observed 2-DG-effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV18 suppression by 2-DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB-8, indicating that Ca2+ a known intracellular "second messenger", is involved in this process. Elevated c-myc and p53 expression appears to be responsible for the time-dependent accumulation of apoptotic cells after prolonged 2-DG treatment. The finding that 2-DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a pecularity of growing tumors, but even may be an essential prerequisite for the maintenance of virus-specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2-DG or other glucose derivatives in the treatment of precancerous and malignant HPV-associated lesions. (C) 1998 Wiley-Liss, Inc.

The non-myelotoxic antitumor drug thaliblastine (thalicarpine, NSC-68075, CAS-5373-42-21) has a novel chemical structure; it is a complex dimeric-type aporphine benzylisoquinoline alkaloid possessing antiproliferative and antitumor activities in experimental and clinical studies. In this study the effect of this drug on the cell cycle progression of ovarian tumor line O-342 and its cisplatin-resistant subline O-342/DDP was evaluated. As assessed by flow cytometric analysis, thaliblastine affected the cell cycle progression. In both lines, a comparable pattern of cell cycle arrest was found. Within the first 5 h of thaliblastine exposure, a G2/M block was observed; thereafter cell-cycle arrest in G1 became prominent, while S-phase-cells finished DNA replication.

While multiple changes are frequently found to be associated with cisplatin resistance in a variety of tumor cell lines, a cause-effect relationship of these alterations with the resistant phenotype has not been established. In order to identify the resistance-relevant determinants, a series of cisplatin-resistant sublines with different degrees of resistance to cisplatin was developed in a human ovarian carcinoma cell line (O-129). Three derived resistant cell lines displayed 2.1-fold (O-129/DDP4, low), 4.1-fold (O-129/DDP8, moderate) and 6.3-fold (O-129/DDP16, high) resistance, respectively, to cisplatin, compared with the sensitive parental line O-129. While the activity of poly(ADP-ribose) polymerase, an enzyme proposed to be involved in DNA repair, was elevated in all three resistant lines, a significant karyotypic change was observed only in the high-resistance line with the karyotype alteration from near diploidy to heteroploidy. The moderate (4.1-fold) and high (6.3-fold) DDP resistance was associated with a slow proliferation rate in drug-free medium, but cellular glutathione level was highly correlated with DDP sensitivity in all four cell lines. Taken together, the present studies establish that while many changes at cellular level can occur with development of cisplatin resistance, only elevation of intracellular glutathione concentration appears to be related to the resistance phenotype in these human ovarian cancer cells.

Lectures and presentations at the 1992 Heidelberg Cytometry Symposium reflected a rapidly growing research field with widespread activities that cover daily routine diagnosis as well as investigations at the molecular level and the diagnosis of genetic alterations. Both flow and image cytometry and their impact on quantitative cytology were backed and combined with new approaches (i.e. magnetic cell sorting) that enable the isolation of rare cells with high purity for cell biological analyses and thus pave the way for new, research fields (i.e. arteriosclerosis). Their combination with the multicolor painting of gene sequences (fluorescent in situ hybridization) represents a further improvement of chromosome quantification and thus the analysis of the topology of cell nuclei.

‘Universal fuser’ clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non‐tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non‐selective conditions. The CAT expressing cells were fused with both non‐tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non‐modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non‐selectable, HPV18‐driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa ×HaCaT cells, but still present in SiHa ×SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non‐tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion‐amplification technique are accompanied by the reactivation of the marker gene in previously CAT‐ SiHa ×HaCaT hybrids. These data strongly suggest that trans‐acting negative regulatory factors derived from the non‐malignant human keratinocytes are responsible for the extinction phenomenon.

In a rat ovarian tumour cell line a 33-fold resistance to cisplatin (O-342/DDP) was developed in vitro by continuous exposure of the parental cell line (O-342) to stepwise increase cisplatin concentration in the culture medium. Both cell lines had a similar growth rate in vitro. Development of resistance was accompanied by a change of the karyotype from heteroploidy in chemosensitive O-342 cells to near diploidy in resistant O-342/DDP cells as shown by chromosome number distribution. This finding was confirmed by measuring cellular DNA content using flow-cytometry analysis. Flow karyotyping showed significant differences in chromosomal DNA contents between both cell lines. Our results suggest that the parent line O-342 consists of at least two subpopulations, a cisplatin-sensitive and a cisplatin-resistant one, corresponding to hyperploidy and near diploidy, respectively. Continuous cisplatin exposure of O-342 cells selectively killed the sensitive fraction, resulting in the karyotypic change observed.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T celts into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis).In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily microdissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

S,gnal transducuon reduced by epidermal growth factor (EGF) vm ,ts receptor revolves two second messengers, chacylglycerol (DG) and momtol msphosphate, both products of phosphandylmo~to|-specdic phosphohpase C wluch seem to pamctpate m the regulauon of cellular prohferauon by EGF We observed that A431 cells and HeL~ cells m response to EGF accumulate substanual amounts of phosphauchc actd (PA), the potenual product of DG kmase However, PA appears earher and stays longer elevated than expected from DG avadabxhty thus rinsing the poss~bd~ty of an EGF-mduced acuvatxon of a phosphohpase D (PLD) In the presence of prm=ry alcohols PLD effects the phosphandyl transfer reacuon producmg PA-alcohol, a reacuon known to be excluszvely ~ by PLD m mtact cells A431 cells and cells prelabeled w~th [14C]-oleate produce m the presence of ethanol (0 5%) or l-butanol (0 2%) substanual amounts of labeled PA-alcohol m response to EGF These results mchcate that acuvauon of PLD had occurred Acuvauon of PLD may x~present a novel mgnal transducing pathway wluch may pamctpate m the regulauon of cell prohferauon by EGF -a hypothes~s presently mvesugated m thts laboratory Supported by the Deutsche Forschungsgememschaft.

N-Nitrosodialkylamines are a unique class of environmental carcinogens which can cause tumors in rodents in a very organ specific manner. The mechanisms for these properties still await clarification. Possible involved parameters could be an organ specific metabolic activation, or the pharmacokinetics of the compounds or of the reactive intermediates. Furthermore, the repair mechanisms, which consequently govern the persistance of specific types of DNA damage may also play an important role. We are presently studying in which manner these individual mechanisms may contribute to organ specific effects of nitrosamines. This is done by assessing their genotoxicity in different organs. In this report, we will present some in vitro and in vivo data on the activities of N-nitrosodimethylamine (NDMA).

This article discusses the results of cytogenetic investigations of a cell line derived from a malignant tumor arising in a benign salivary gland pleomorphic adenoma. Initial karyotypic studies became possible with cells of the second in vitro passage and revealed the existence of hypertetraploidy. Furthermore, a marked polysomy 7 and a translocation involving 12q13-15, a breakpoint also frequently seen in benign pleomorphic adenomas, were noteworthy. During long term culture, the number of chromosomes per cell decreased as well as the number of copies of chromosome 7. Cell tumorigenicity was proved by heterotransplantation to nude mice. The resulting tumors were karyotyped again. No significant changes of the chromosome number or of the degree of polysomy 7 were found compared to the cells before heterotransplantation. In contrast, the cells from the nude mice tumors showed a remarkable number of different isochromosomes probably indicating an unknown factor supporting the generation of isochromosomes. The consistent presence of the t(12;?)(q13-15;?) makes the cell line well suited for molecular studies of this breakpoint region.

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.